JP5819954B2 - Novel calcium salts of compounds as anti-inflammatory, immunomodulatory and antiproliferative agents - Google Patents

Description

  The present invention relates to novel salts of compounds used as anti-inflammatory agents, immunomodulators and anti-proliferative agents. In particular, the present invention relates to novel salts of compounds that inhibit dihydroorotate dehydrogenase (DHODH), processes for their preparation, pharmaceutical compositions containing them, and uses for the treatment and prevention of diseases, in particular dihydroorotate It relates to the use in diseases that are advantageous in inhibiting dehydrogenase (DHODH).

  Rheumatoid arthritis (RA) is a very common disease, especially in older people. Treatment with conventional medicines such as non-steroidal anti-inflammatory drugs is not satisfactory. Development of new drugs for the treatment of RA is urgently needed in terms of increasing aging in the population, especially in the developing western countries or Japan.

  International Publication No. WO 2003/006425 describes certain compounds that have been reported to be useful for the treatment or prevention of diseases that are advantageous in inhibiting dihydroorotate dehydrogenase (DHODH). Yes. However, specific salts of the present invention are not disclosed.

  International Publication No. WO 99/38846 and European Patent No. EP 0 646 578 disclose compounds that are reported to be useful in the treatment of RA.

  A drug for rheumatoid arthritis having a novel mechanism of action, leflunomide, was recently sold by Avenchus under the trade name ARAVA [EP 780128, WO 97/34600]. Leflunomide has immunomodulation and anti-inflammatory properties [EP 217206, DE 2524929]. The mechanism of action is based on the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis.

  De Julian-Ortiz (J. Med. Chem. 1999, 42, 3308-3314) describes certain potent anti-herpes compounds having a cyclopentenoic acid moiety.

  DE 33 46 814 A1 describes carboxylic acid amide derivatives for the treatment, prevention and alleviation of diseases associated with impaired brain function and the symptoms caused thereby.

  In the body, DHODH catalyzes the synthesis of pyrimidines required for cell growth. Inhibition of DHODH inhibits pathologically fast-growing cells, whereas cells that grow at normal rates can obtain their required pyrimidine bases from normal metabolic cycles. The most important cell type for the immune response, lymphocytes, uses pyrimidine synthesis exclusively for its proliferation and reacts particularly sensitively to DHODH inhibition. Substances that inhibit the proliferation of lymphocytes are important drugs for the treatment of autoimmune diseases.

  The DHODH inhibitor leflunomide (ARAVA) is the first drug (leflunomides) of this type of compound for the treatment of rheumatoid arthritis. WO 99/45926 is a further document disclosing compounds that act as inhibitors of DHODH.

  JP-A-50-121428 discloses N-substituted cyclopentene-1,2-dicarboxylic acid monoamides and their synthesis as herbicides. For example, N- (4-chlorophenyl) -1-cyclopentene-1,2-dicarboxylic acid monoamide is prepared by reacting 1-cyclopentene-1,2-dicarboxylic anhydride with 4-chloroaniline.

Journal of Med. Chemistry, 1999, Vol. 42, pages 3308-3314 describes virtual combinatorial synthesis and computational screening of new potent anti-herpes compounds. Table 3 on page 3313 contains 2- (2,3-difluorophenylcarbamoyl) -1-cyclopentene-1-carboxylic acid, 2- (2,6-difluorophenylcarbamoyl) -1-cyclopentene-1-carboxylic acid , And 2- (2,3,4-trifluorophenyl-carbamoyl) -1-cyclopentene-1-carboxylic acid, the experimental results on IC ₅₀ and cytotoxicity are described.

  DE 3346814 and US 4661630 disclose carboxylic acid amides. These compounds are useful for diseases associated with brain disorders and have anti-ulcer, anti-asthma, anti-inflammatory, and low cholesterol activity.

  EP 0097056, JP 55157547, DE 2851379 and DE 2921002 describe tetrahydrophthalamic acid derivatives.

  It is an object of the present invention to provide another effective agent, in particular another effective agent, particularly in the form of its calcium salt, which can be used for the treatment of diseases requiring the inhibition of DHODH .

  Specifically, compounds of general formula (I) shown herein below, such as 2- (3-fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid ( It has previously been shown that INN Bidofludims has shown excellent anti-inflammatory activity and its usefulness in oral therapy for the treatment of autologous diseases such as rheumatoid arthritis or inflammatory bowel disease. Has been issued. However, the solubility of the above compounds in aqueous media is less than 1 mg / ml at neutral pH.

  Compound solubility is an important feature in drug discovery because it serves as a starting point for formulation development. Furthermore, after oral administration, the absorption of the drug from the intestine to the circulation is at least partially dependent on its solubility. Only the dissolved substance is expected so that increased solubility is expected to result in better uptake of the compound in the gastrointestinal tract, i.e. better oral bioavailability and improved pharmacokinetic properties Is absorbed. In order to improve its use as a pharmaceutical compound for oral application, it is desirable to provide compounds with high bioavailability.

  Accordingly, the inventors have made efforts to increase the solubility of a compound in an aqueous medium and thus its bioavailability. This has motivated studies to develop new salt forms of the compounds, resulting in unexpectedly significantly improved physicochemical properties.

  Calcium salts of compounds of formula (I) as described herein below, for example calcium salts of 2- (3-fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid It has been found that it exhibits favorable physicochemical properties, such as improved water solubility, while maintaining good long-term stability. Furthermore, it has been found that not all of the salts of the compound generally increase the water solubility to the same extent; in fact, the solubility of the tested salts is all higher than the free acid, but they Are significantly different from each other. More importantly, the calcium salt exhibits a significantly higher bioavailability compared to other salts or free acids.

  Thus, a new class of calcium salts of compounds having an inhibitory effect on DHODH, particularly human DHODH, has been found.

  Accordingly, the present invention provides a compound of the general formula (I):

[Where:
X is selected from the group consisting of CH ₂ , S and O;
D is O or S;
R ⁸ is hydrogen or alkyl, preferably hydrogen or methyl;
E is an optionally substituted phenylene group;
Y is a monocyclic or bicyclic substituted or unsubstituted 6-9 membered ring system containing one or more heteroatoms selected from N and S and containing at least one aromatic ring, preferably Y is substituted or unsubstituted phenyl;
n is 0 or 1, preferably 0; and
q is 0 or 1, preferably 0. ]
Or a hydrate thereof (except for a compound where q = 0, Y = unsubstituted phenyl, and E = unsubstituted phenylene). ]
About.

FIG. 1 shows the reduction in human lymphocyte proliferation caused by 2- (biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid used at a concentration of 100 μM. FIG. 2 shows a comparison of the plasma level of bidofludimus free acid (black spot) and its calcium salt (white spot) in rats after a single oral dose of 10 m // kg body weight. FIG. 3 shows Raman spectra of Ca salt (upper figure) and free acid (lower figure). FIG. 4 shows an optical micrograph having cross-polarized light of a bidfurdymium Ca salt. FIG. 5 shows the PXRD of Bidfludymus Ca salt. FIG. 6 shows the PXRD of Bidofludimus free acid.

  E is preferably a phenyl group or a phenyl group substituted with one or more groups independently selected from halogen, nitro and alkoxy; more preferably, E is one fluorine, one chlorine atom , A phenylene group substituted by one methoxy group or four fluorine atoms. Even more preferably, E is a phenylene group substituted with 1 or 4 fluorine atoms, and even more preferably a phenylene group substituted with 1 fluorine atom.

  Y is preferably an optionally substituted phenyl, pyridine, or benzothiophene group. More preferably, Y is unsubstituted phenyl or a phenyl group substituted with one or more groups independently selected from halogen, alkyl, alkoxy, haloalkoxy, haloalkyl and CN. Even more preferably, Y is a phenyl group substituted with one or more groups independently selected from fluorine, chlorine, CN, methoxy, ethoxy, trifluoromethyl and trifluoromethoxy. Even more preferably, Y is one or more groups independently selected from methoxy and trifluoromethoxy, and even more preferably a phenyl group substituted with methoxy.

  Preferably, the salt of the present invention is selected from N-methyl-D-glucamine (NMG) salt and diethylamine (DEA) salt. In addition to its high solubility, the salt exhibits desirable properties related to hygroscopicity, which is desirable in formulating the compound into a medicine.

  Unless otherwise stated, alkyl groups are preferably straight or branched, saturated with 1 to 6 carbon atoms, preferably methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, A hexyl group, preferably a methyl, ethyl, propyl, isopropyl or t-butyl group, even more preferably a methyl or ethyl group, still more preferably a methyl group.

  Alkoxy refers to an O-alkyl group in which the alkyl group is defined above.

A haloalkyl group refers to an alkyl group that is substituted with one or more halogen atoms, as defined above; trifluoromethyl is preferred. A haloalkyl group refers to an alkoxy group substituted by one or more halogen atoms, as defined above; OCF ₃ is preferred.

  Halogen is preferably chlorine, bromine, fluorine or iodine, preferably fluorine, chlorine or bromine, more preferably fluorine.

  The present invention also provides a pharmaceutical composition comprising a calcium salt of a compound of formula (I) as described above together with a pharmaceutically acceptable diluent or carrier thereof.

  In another aspect, the invention also treats or prevents a condition beneficial to inhibit dihydroorotate dehydrogenase (DHODH) comprising administering an effective amount of a calcium salt of a compound of formula (I) as described above. Provide a way for.

  The present invention also relates to the use of a calcium salt of a compound of formula (I) as described above for the manufacture of a medicament for the prevention and treatment of diseases where inhibition of pyrimidine biosynthesis is advantageous.

  The present invention also provides that the crystals obtained from the salt have a specific stoichiometric amount or a substoichiometric amount of water, such as 0.5, 1, or per molecule of a compound of formula (I) as described herein. Also encompassed are hydrates of the salts of the present invention, which are specified to contain two water molecules.

  Further preferred aspects of the present invention are summarized in the following items [1] to [8].

[1] Calcium (Ca), diethylamine (DEA), N-methyl-D-glucamine (NMG), lithium (Li), zinc (Zn), L-arginine, 4- (2-hydroxyethyl) morpholine (HEM) A compound of formula (1a) having a base selected from the group consisting of L-lysine (LYS), choline (CHO) and ammonia (NH ₃ ):

Salt.

[2] 2- (3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) having a base selected from calcium (Ca), diethylamine (DEA), and N-methyl-D-glucamine (NMG) -The compound of item [1], which is a salt of cyclopent-1-enecarboxylic acid.
[3] A pharmaceutical composition comprising a compound defined in any one of items [1] to [2] and a pharmaceutically acceptable diluent or carrier thereof.
[4] A compound according to items [1] to [2] for use as a medicament.
[5] Items [1] to [2] in the manufacture of a medicament for use in the treatment of a disease or medical condition in which dihydroorotate dehydrogenase and / or interleukin 17 (IL-17) is effective Use of a compound of formula (1a) as defined in any of the above.
[6] The use of item [5], wherein the disease or symptom is an autoimmune disease.
[7] The autoimmune disease is ankylosing spondylitis, autoimmune thyroiditis, Syracic disease, Graves' disease, inflammatory bowel disease (Coulomb disease, ulcerative colitis), type 1 diabetes, systemic lupus erythematosus, multiple The use of paragraph [6], selected from the group consisting of sclerosis, vitiligo, osteoarthritis, psoriasis, psoriatic arthritis, and rheumatoid arthritis.
[8] Use of a compound of formula (1a) as defined in any of items [1] to [2] in the manufacture of a medicament for use in the treatment of any form of neoplasm.

  In addition, the present invention provides methods for preparing the compounds of the present invention, for example the desired amides of formula (I) and the calcium salts thereof described above.

  A first method for the synthesis of amides of formula (I) comprises reacting an acid anhydride of formula (II) with an amine of formula (III).

The second method of the present invention for preparing the compound of formula (I) is to prepare an amine of formula (IV) and an arylboric acid of general formula (V): (HO) ₂ BE- [D- (CHR ³ ) _n ] Including reacting with _q- Y [MP Winters, Tetrahedron Lett, 39, (1998), 2933-2936].

  A third method of the invention for preparing a compound of formula (I) comprises reacting a halogen derivative of formula (VI) with an aryl boric acid of general formula (VII) [NE Leadbeater, SM Resouly, Tetrahedron , 55, 1999, 11889-11894]. Q is a halogen group such as chlorine, bromine, fluorine or iodine, preferably bromine.

  In the structures of Formulas II-VII described herein, residues X, E, D, Y, Rg and variables n and q have the meanings defined herein for Formula I.

Preferably, the salt of the invention is a calcium salt derived from a compound selected from the group comprising the following compounds 1 to 76:
1. 2-((4- (benzyloxy) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
13. 2-(((2'-Fluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
14. 2-(((2'-Chloro- [1,1-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
15. 2-((2'-Methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
16. 2-((4-((2,6-Difluorobenzoyl) oxy) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
17. 2-(((2'-Ethoxy-3-fluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
18. 2-((2-Chloro-3'-cyano- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
19. 2-((2-Chloro-4'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
22. 2-((3-Chloro-4- (6-methoxypyridin-3-yl) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
23. 2-((4'-Methoxy-2-methyl- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
24. 2-((3,5-Dibromo-4-((2,5-difluorobenzyl) oxy) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
25. 2-((3,5-Dibromo-4-((3,4-difluorobenzyl) oxy) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
26. 2-((3-Fluoro-3'-methoxy- [1,1-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
27. 2-((3,3'-difluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
29. 4-((4- (Benzyloxy) -3,5-dibromophenyl) carbamoyl) -2,5-dihydrothiophene-3-carboxylic acid
30. 2-((3,4 ', 5-Trifluoro-3'-methyl- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
31. 2-((3,5-Difluoro-2'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
32. 2-((2'-Methoxy-3-nitro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
33. 2-((3-Methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
34. 2-((2'-Chloro-3-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
35. 2-((3-Methoxy-3 '-(trifluoromethoxy)-[1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
36. 2-((2'-Fluoro-3-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
37. 2-((2,3,4'-Trimethoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
38. 2-((3'-Ethoxy-3-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid carbamoyl
39. 2-((3,3'-Dimethoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
40. 2-((3,5-Dibromo-4-((2-chloro-6-fluorobenzoyl) oxy) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
41. 4-((2'-Chloro- [1,1'-biphenyl] -4-yl) carbamoyl) -2,5-dihydrothiophene-3-carboxylic acid
42. 2-((4- (m-Tolylthio) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
43. 2-((3 '-(Trifluoromethoxy)-[1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
44. 2-((4- (Benzo [b] thiophen-2-yl) phenyl) carbamoyl) cyclopent-1-enecarboxylic acid
45. 2-((4- (Benzo [b] thiophen-2-yl) -2-fluorophenyl) carbamoyl) cyclopent-1-enecarboxylic acid
46. 2-(((3'-Ethoxy-3-fluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
47. 4-((3,5-Difluoro-2'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) -2,5-dihydrofuran-3-carboxylic acid
50. 2-((4-Phenoxyphenyl) carbamoyl) cyclopent-1-enecarboxylic acid
52. 4-((3,5-Dibromo-4-((2-chloro-6-fluorobenzoyl) oxy) phenyl) carbamoyl) -2,5-dihydrothiophene-3-carboxylic acid
53. 2-((3-Chloro-2'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
54. 2-((2-Chloro-2'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
56. 2-((2,3,5,6-Tetrafluoro-2'-methoxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
57. 2-((2'-Methoxy-3-methyl- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
58. 2-((3,5-Dichloro-2'-methoxy- [1,1'-biphenyl] -4-yL) carbamoyl) cyclopent-1-enecarboxylic acid
62. 2-((2'-Ethoxy-3,5-difluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
63. 2-((3'-Ethoxy-3,5-difluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
64. 2-((3,5-Difluoro-3 '-(trifluoromethoxy)-[1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
65. 2-((2'-Chloro-3,5-difluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
66. 2-((2 ', 3,5-Trifluoro- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
67. 2-((3,5-Difluoro-2 ', 4'-dioxy- [1,1'-biphenyl] -4-yl) carbamoyl) cyclopent-1-enecarboxylic acid
68. 2-((3-Chloro-4-((2- (trifluoromethyl) benzyl) oxy) phenyl) carbamoyl cyclopent-1-enecarboxylic acid
69. 2-((3-Chloro-4-((2-chloro-6-fluorobenzyl) oxy) phenyl) carbamoyl cyclopent-1-enecarboxylic acid
72. 2-((4- (Benzyloxy) -3-chlorophenyl) carbamoyl) cyclopent-1-enecarboxylic acid
74. 2-((3-Chloro-4-((2-fluorobenzyl) oxy) phenyl) carbamoyl cyclopent-1-enecarboxylic acid
76. 2-(((3-Fluoro-2′-methoxy- [1,1′-biphenyl] -4-yl) oxy) carbonyl) cyclopent-1-enecarboxylic acid.

  Preferably, the calcium salt of the present invention is a calcium salt as described above wherein the compound of formula (I) is selected from the group comprising compounds shown in Table 1 below.

  The compound of formula (I) can be obtained by various methods including the method described in JP-A-50-121428. In a preferred embodiment of the method of the invention, the following two synthetic methods are used.

  Method 1: In the first step, the cycloalkene-1,2-dicarboxylic acid is prepared by the corresponding α, α described by RN Mc Donald and RR Reitz, J. Org. Chem. 37, (1972) 2418-2422. Obtained from '-dibromoalkanedicarboxylic acid. Cyclopentene-1,2-dicarboxylic acid can also be obtained in large quantities from pimelic acid [DC Owsley und J J. Bloomfield, Org. Prep. Proc. Int. 3, (1971) 61-70; R. Willstatter, J. Chem. Soc. (1926), 655-663].

  Dicarboxylic acids substituted within or on a ring system are generally synthesized by cyanohydrin synthesis [Shwu-Jhan Lee et.al., Bull. Inst. Chem, Academia Sinica Number 40, (1993), 1- 10, or BR Baker at al., J. Org. Chem. 13, 1948, 123-133; and BR Baker at al., J. Org. Chem. 12, 1947, 328-332; LA Paquette et. Al, J. Am. Chem. Soc. 97, (1975), 6124-6134].

  The dicarboxylic acid is then converted to the corresponding acid anhydride by reacting it with acetic anhydride [P. Singh and S.M. Weinreb, Tetrahedron 32, (1976), 2379-2380].

  Other methods for preparing different acid anhydrides of formula (II) are described in VA Montero et al., J. Org. Chem. 54, (1989), 3664-3667; P. ten Haken, J. Heterocycl. Chem. 7, (1970), 1211-1213; K. Alder, H. Holzrichter, J. Lieb. Annalen d. Chem. 524, (1936), 145-180; Alder, E. Windemuth, J. Lieb. Annalen d Chem. 543, (1940), 56-78; and W. Flaig, J. Lieb. Annalen d. Chem. 568, (1950), 1-33.

  These anhydrides can then be reacted with the corresponding amine to give the desired amide of formula (I). This reaction can be performed by using the reaction conditions described in JV de Julian Ortiz et al., J. Med. Chem. 42, (1 99), 3308 (predetermined route A in Example 1) or by 4-dimethylamino This can be done by the use of pyridine (predetermined route B of Example 1).

  Method 2: Amides of formula (I) can also be synthesized by reacting amines of formula (IV) with arylboric acids of general formula (V) [MP Winters, Tetrahedron Lett., 39, (1998), 2933 -2936].

  Biarylanilines can generally be synthesized by palladium coupling [GW Kabalka et al., Chem. Commun., (2001), 775; A. Demeter, Tetrahedron Lett. 38; (1997), 5219-5222; V. Snieckus, Chem. Commun. 22, (1999), 2259-2260].

  Method 3: Amides of formula (I) can also be synthesized by reacting halogen derivatives of formula (VI) with arylboric acids of general formula (VII) [NE Leadbeater, SM Resouly, Tetrahedron, 55, 1999, 11889- 11894].

The calcium salts of the present invention can be used for a variety of human and animal diseases, preferably human diseases, where inhibition of pyrimidine metabolism is advantageous. Such diseases are the following:
-Fibrosis, uveitis, rhinitis, asthma or arthropathy, in particular joints;
-All forms of rheumatism;
-Acute immunological events and disorders such as sepsis, septic shock, endotoxin shock, Gram-negative bacterial sepsis, toxic shock syndrome, acute respiratory failure syndrome, stroke, reperfusion injury, CNS injury, significant forms of allergy Vs. host graft reaction, Alzheimer's or fever, restenosis, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption disease. Immunological events also include the desired regulation and suppression of the immune system;
All types of autoimmune diseases, especially rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, multiple sclerosis, insulin-dependent and non-insulin-dependent diabetes, and lupus erythematosus, Ulcerative colitis, Morbus Crohn's disease, inflammatory bowel disease, and other chronic inflammation, chronic diarrhea;
-Dermatological disorders such as psoriasis;
-Progressive retinal atrophy;
-All types of infections, including opportunistic infections.

  The calcium salt of the present invention and a medicament prepared using the same are generally used for the treatment of cell proliferative diseases, immune diseases and symptoms (for example, inflammatory diseases, neuroimmune diseases, autoimmune diseases and the like) or Useful for prevention.

  The calcium salts of the present invention are also useful for the development of immunomodulatory and anti-inflammatory drugs, and more generally for the treatment of diseases where inhibition of pyrimidine biosynthesis is advantageous. The salts of the present invention are also useful for the treatment of diseases caused by malignant cell proliferation, eg all forms of blood and solid tumors. Thus, the compounds of the present invention and medicaments prepared using them are generally used to regulate cell activation, cell proliferation, cell survival, cell differentiation, cell cycle, cell maturation and cell death, or saccharides Useful for inducing systemic changes in metabolism, such as changes in lipid or protein metabolism. They also include blood cell growth and development (pre-hematopoietic effect) after cell depletion or destruction caused by, for example, toxic substances, radiation, immunotherapy, growth defects, malnutrition, malabsorption, immune dysregulation, anemia, etc. It can be used to support cell development or provide therapeutic control of tissue development and disruption, therapeutic modification of cell and tissue maintenance, and blood cell homeostasis.

  These diseases and symptoms include blood cancer (eg, leukemia, lymphoma, myeloma) or solid tumor (eg, breast cancer, prostate cancer, liver cancer, bladder cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, genitourinary organs) Cancer, gastrointestinal cancer, skin cancer, pancreatic cancer, brain tumor, uterine cancer, colon cancer, head and neck cancer, and ovarian cancer, melanoma, astrocytoma, small cell lung cancer, glioma, basal cell carcinoma and squamous cell carcinoma , Sarcomas such as Kaposi sarcoma and osteosarcoma), treatment of disorders associated with T-cells such as but not limited to aplastic anemia, DiGeorge syndrome, Graves' disease.

  Leflunomide was previously found to inhibit HCMV replication in cell culture. Ocular herpes is the most common cause of infectious blindness in developing countries. In the United States alone, there are about 50,000 cases per year, 90% of which are recurrent primary infections. Recurrence is treated with antiviral agents and corticosteroids. Other herpesviruses of cytomegalovirus are common causes of retinal damage and blindness in patients with AIDS. The compounds of the invention can be used alone or in combination with other antiviral agents such as ganciclovir and foscarnet to treat such diseases.

  Moreover, the calcium salt of the present invention can be further used in diseases caused by protozoal infections in humans and animals. Such veterinarians and human pathogenic protozoa are preferably intracellular activity parasites of the apicomplexa or fleshly flagellate, in particular trypanosoma, plasmodia, leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, amebiasis, coccidia and Trichomonadia. These active substances or corresponding drugs are caused by tropical malaria caused by Plasmodium falciparum, Plasmodium falciparum or O. falciparum malaria caused by Plasmodium falciparum, and by Plasmodium falciparum It is particularly suitable for the treatment of heat radiation malaria. These substances are also found in Toxoplasma gondii caused by Toxoplasma gondii, for example, coccidiosis caused by Isospora belli, Intestinal Sarcosporidiosis caused by porcine granulocysts ), Dysentery caused by dysentery amoeba, cryptosporidiosis caused by Cryptosporidium parvum, Chagas disease caused by Trypanosoma cruzi, trypanosoma brucei rhodesiens or sleep illness caused by Trypanosoma brucei rhodesiens or gambiense Suitable for the treatment of not only skin and internal organs, but also other forms of Leishmaniosis. These substances can also be found in Theileria parva, a pathogen that causes east coast fever in cattle, Trypanosoma congolense congolense, a pathogen that causes livestock Nagaana disease in Africa, or Trypanosoma vivax vivax, Trypanosoma brucei brucei, Trypanosoma brucei evansi, which causes surah disease, Babesibies, the causal agent that causes Texas fever in cattle and buffalo , Not only babesiosis in European cattle, but also circustiosis, the causative agent that causes Sarcocystiotis in Babesia bovis, sheep, cattle, and pigs, which are causative agents that cause babesiosis in dogs, cats, and sheep Sarcocystis ovicanis and Sarcocystis ovifelis, Cryptosporidia that causes cryptosporidiosis in cattle and birds, rabbits, cattle, sheep, goats, pigs and birds, especially chickens It is suitable for the treatment of animals infected by veterinary pathogenic protozoa, such as Eimeria and Isospora species, the pathogens that cause turkey coccidiosis. The use of the compounds of the present invention is particularly preferred for the treatment of coccidiosis or malaria infection, or the manufacture of drugs or feeds for disease treatment. Such treatment can be used prophylactically or curatively. In the treatment of malaria, the compounds of the present invention can be used in combination with other antimalarial agents. The use of the compounds of the invention is particularly preferred for the treatment of coccidiosis or malaria infection or for the preparation of drugs or feed ingredients for the treatment of these diseases. This treatment may be prophylactic or curative. In the treatment of malaria, the compounds of the invention may be combined with other antimalarial agents.

  The calcium salts of the present invention can further be used for viral infections and other infections caused by, for example, Pneumocystis carini.

  Preferably, the disease or condition to be treated or prevented by the calcium salt of the present invention is selected from the group comprising graft versus host reaction, rheumatoid arthritis, multiple sclerosis, lupus erythematosus, inflammatory bowel disease and psoriasis Is done.

  The calcium salts of the compounds of formula (I) according to the present invention allow for intestinal or parenteral use as therapeutic agents themselves, as mixtures with others, or in conventional pharmaceutically harmless shaping In the form of a pharmaceutical composition comprising an effective amount of at least one calcium salt of the above compound of formula (I) of the present invention as an active ingredient together with agents and additives, animals, preferably mammals, and especially humans, dogs And to chickens.

  The therapeutic can be administered orally, for example as pills, tablets, coated tablets, sugar-coated tablets, hard and soft gelatin capsules, solutions, syrups, emulsions or suspensions, or aerosol mixtures thereof. However, administration can also be effected rectally, for example in the form of suppositories, or parenterally, for example in the form of injections or infusions, or transdermally, for example in the form of ointments, creams or tinctures.

  In addition to the salt of the active compound of formula (I), the pharmaceutical composition may further comprise customary, normally inert carrier materials or excipients. Accordingly, pharmaceutical preparations are made of additives such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavoring or fragrances, buffer substances And may further include a solvent or solubilizer or agent to achieve the depot effect, as well as salts, coatings or antioxidants to alter the osmotic pressure. They may contain the above-mentioned salts of two or more compounds of formula (I) and other therapeutically active substances.

  The salts according to the invention can therefore be used alone or in combination with other active compounds, for example already known medicaments for the treatment of the above diseases, thereby amplifying the effect in the latter case Preferred additives are noted. Suitable amounts to be administered to humans are in the range of 5 to 500 mg.

  To prepare the pharmaceutical preparations, pharmaceutically inert inorganic or organic excipients can be used. For preparing pills, tablets, coated tablets and hard gelatin capsules, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof, and the like can be used. Soft gelatin capsules and suppository excipients are, for example, lipids, waxes, semi-solid and liquid polyols, natural or hardened oils, and the like. Suitable excipients for the production of solutions and syrups are, for example, water, sucrose, invert sugar, glucose, polyols and the like. Suitable excipients for the manufacture of injectable solutions are, for example, water, alcohols, glycerol, polyols or vegetable oils.

  The dosage can vary within wide limits and will be adapted to the individual symptoms in each case. For the above uses the appropriate dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired. In general, however, satisfactory results are achieved at dosage rates of about 1-100 mg kg animal body weight, preferably 1-50 mg / kg. Suitable dosage rates for large mammals such as humans are on the order of about 10 mg to 3 g / day, conveniently in divided doses once, 2 to 4 times daily or in sustained release form Is administered.

  In general, a daily dose of about 10 mg to 5000 mg, preferably 50 to 500 mg per person is suitable for oral administration, which is the preferred form of administration of the present invention. The daily dose is in the same range for other dosage forms.

  The invention is further illustrated by the following non-limiting examples. The data presented for the specific compounds listed in Table 1 above also relate to specific examples of the present invention.

Experiment / Equipment settings
¹ H-NMR: ¹ H-NMR spectra were recorded on a Bruker DPX300 spectrometer with a proton frequency of 300.13 MHz, 30 ° excitation pulse and accumulated a recycling delay of 1s.16 scans.
D ₂ O; MeOD or d ₆ -DMSO was used as the solvent.
Solubility: Solubility was measured by adding 0.05 mL portions of solvent dropwise to a suspension of 10 mg sample in 0.05 mL of solvent. If the substance does not dissolve upon addition of a total of 10 mL of solvent, solubility is indicated as less than 1 mg / mL.

DSC: Differential scanning calorimetry was performed using a Perkin Elmer DSC-7 apparatus (closed gold sample pan under N ₂ atmosphere). The sample was heated to the melting point at a rate of 1 OK / min, then cooled (cooling rate 200 K / min) and then reheated at a rate of 1 OK / min.

  DVS (SMS): DVS-1 water vapor absorption analyzer from Surface Measurement Systems Ltd. The sample was placed on a platinum sample pan and equilibrated at a given relative humidity (r.h.), usually 50% r.h. A pre-established humidity program was then started with a scan rate of 5% r.h. charge / hour. 1st step: 50% rh to 0% rh (50-95% rh in the case of a possible hydrate as starting material), 2nd step: 0% rh to 95% rh (possible as starting material) In the case of sexual hydrates, 95-0% rh).

FT-Raman spectroscopy: FT-Raman spectra were recorded on a Bruker RFS 100 FT-Raman instrument with a near-infrared Nd: YAG laser operating at 1064 nm and a liquid nitrogen cooled germanium detector. For each sample, a minimum of 64 scans with a resolution of 2 cm ^-1 were integrated. A 300 mW apparent laser was used. FT-Raman data is found in the region of 350-100 cm ^-1 . Below 100 cm- ¹ , the data is not reliable due to the Rayleigh wave filter cutoff.

  Optical microscope: Leitz Orthoplan 110680 microscope with Leica DFC280 camera and IM50 v.5 image acquisition software. Images were recorded at 4x, 1Ox or 25x magnification with or without crossed polarizers.

  Powder X-ray diffraction: Bruker D8; Copper Ka radiation, 40 kV / 40 mA; LynxEye detector, 0.02 ° 2Θ step time, 37 s step time. Sample preparation: Samples were generally measured without specific treatment other than the application of light pressure to obtain a flat surface. A silicon single crystal sample holder was used (0.1, 0.5 or 1 mm depth). The sample was rotated during the measurement.

Raman spectroscopy: Renishaw in Via Reflex Raman System. Stabilized diode laser with 785 nm excitation and NIR augmented Peltier-cooled CCD camera as detector. Measurements were made with a long working distance objective 20x. Wavelength range 2000-100 cm ^-1 , 10 s detection time, 3 cumulative / spectrum.

  Solvent: For all experiments, Fluka, Merck or ABCR analytical grade solvents were used.

  TG-FTIR: Netzsch Thermo-Microbalance TG 209 connected to Braker FTIR Spectrometer Vector 22 or IFS 28 (sample pan with pinhole, N2 atmosphere, 10 ° C / min heating rate, 25 ° C to 350 ° C range) Gravimetric analysis was performed.

  HPLC: HPLC consists of solvent rack, vacuum degasser, binary pump (mikro), static mixer (500 μl), autosampler, 25 μl sample loop, 100 μl syringe, column oven, and DAD detector set up for UV analysis ( Dionex UltiMate (registered trademark) 3000 liquid chromatograph including a semi-micro measurement cell). Data analysis was performed with Chromeleon 6.80 SP3. Compounds were separated on a Phenomenex Onyx ™ Monlithic C18 50x2 mm column at 30 ° C. The injection volume was 2 μl and the detection wavelength was 305 nm. A gradient of 0.1% formic acid in HPLC grade water / acetonitrile was used as the mobile phase, starting at a concentration of 5% acetonitrile. The starting concentration is maintained for 1 minute, then linearly tilted to a concentration of 95% acetonitrile over 2 minutes, maintained at 95% acetonitrile for 0.7 minutes, then returned to 5% acetonitrile within 0.1 minutes and held constant for 0.7 minutes. Maintain and re-equilibrate the column. The mobile phase flow rate was 1.5 ml / min.

Example 1.
The synthesis of compounds of formula (I) is described in detail in WO 2003/0006425, which is hereby incorporated by reference.

Example 2. Inhibition assay of DHODH activity The standard assay mixture contained 50 μM decyclo ubitinone, 100 μM dihydroorho salt, 60 μm 2,6-dichloroindophenol, and 20 mU DHODH. The volumetric activity of the recombinant enzyme used was 30 U / ml. Measurements were made with 50 mM TrisHCl (150 mM KC1, 0,1% Triton X-100, pH 8,0) at 30 ° C. in a final volume of 1 ml. The components were mixed and the reaction was started by adding dihydroorotate. The progress of the reaction was followed by spectroscopically measuring the decrease in absorption at 600 nm for 2 minutes.

Inhibition studies were performed in a standard assay with additional changes in inhibitor. At least 5 different inhibitor concentrations were applied for determination of IC ₅₀ values (inhibitor concentration required for 50% inhibition).

  These studies were performed by recombinant humans and recombinant murine DHODH provided by Prof. M. Loffler, Marburg, Germany [M. Loffler, Chem. Biol. Interact. 124, (2000), 61-76]. did.

  As a reference, the active metabolite of leflunomide A77-1726 (compound 12) was used [J. Jockel et al. Biochemical Pharmacology 56 (1998), 1053-1060].

The results of the inhibition assay are shown in Table 1. The compounds used in the preparation of the salts of the present invention will not only have comparable or better inhibitory activity to the human enzyme than the active metabolite of leflunomide, but will also have a higher specificity for the human enzyme. This is evident from a comparison of IC ₅₀ values.

Example 3: Human T-Cell Proliferation Assay Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers and transferred to PMRI 1640 cell culture medium containing 10% dialyzed fetal bovine serum. 80,000 cells / well were pipetted into 96-well plates and phytohemagglutinin (PHA) was added to phosphate buffered saline to a final concentration of 20 μg / ml to stimulate T-cell proliferation. Bidfludim was added to dimethyl sulfoxide (DMSO, final concentration: 0.1 Vol%) to a final concentration of 20 nM to 50 μM. After 48 hours of incubation, cell proliferation was quantified using a “cell proliferation ELISA BrdU” (Roche) according to the manufacturer's instructions. Half maximum inhibition (IC ₅₀ ) was calculated using a 4-parameter sigmoid curve. T-cell proliferation was inhibited by bidfludim, with an IC ₅₀ of 4.1 μM (see FIG. 1).

Example 4: Preparation of calcium salt
300.4 mg of bidofludim free acid was dissolved in 18 mL DCM / MeOH (3: 1). 31.5 mg of calcium was suspended in 3 mL DCM / MeOH (3: 1); this was slowly added to the Bidfludimus free acid solution. This light suspension was stirred at 25 ° C. overnight. The solvent was partially concentrated at 25 ° C. under a nitrogen stream. A high viscosity light yellow suspension was observed. The solid was collected by filtration and washed with DCM / MeOH (3: 1). The solid was dried under reduced pressure at 25 ° C. for 15 minutes. The material was found to be crystalline using the method described below.

  Sample B) About 1/5 of the solid prepared from Sample A was suspended in 500 μl of 1,4-dioxane / TBME (tert-butyl methyl ether) (1: 1) and stirred at 25 ° C. for 3 days. A partially crystalline material (sample B) was obtained.

  From the elemental analysis, the ratio of nitrogen to fluorine was calculated. Elemental composition analysis is essentially consistent with hemicalcium salt.

The Raman spectrum of the newly formed compound demonstrated a difference from that of the free acid (see Figure 3 for the two spectra). Note that the Raman spectrum is not simply a superposition of the free acid, previous salt, and solvent spectra, eg, the Raman spectrum in which a new or shifted peak is observed may fall under the salt. . However, it cannot be determined from the Raman spectrum alone whether crystal salt formation has occurred. The peak shift is also principally due to free acid and complexation of the previous salt as an amorphous product, polymorphs, purity, or decay products of either the free acid or the previous salt. Therefore, the entire molecular structure was confirmed by ¹ H-NMR.

  In addition, the powder X-ray diffraction shown in FIG. 5 shows that a crystalline material was obtained, but has a pattern different from that of the free acid (see FIG. 6). The crystals were visualized using optical microscopy (Figure 4), DSC (Differential Scanning Calorimetry) showed a melting point of about 155 ° C, thermogravimetric coupled with TG-FTIR (Fourier transform infrared spectroscopy / mass spectrometry) Analyzer) probably formed methanol solvates and hydrates, and the dynamic water vapor adsorption / desorption measuring device gave 0.3% water uptake at about 85% rh and 0.4% at 95% rh (reversible) .

Example 5: Solubility test of this compound Bidofludimus free acid (2- (3-fluoro-3'-methoxybiphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid), its potassium salt and its calcium salt Was tested for water solubility. The principle of the method was based on the OECD Guideline 105 “Water-soluble” for testing chemicals. 2 mg of experimental compound was weighed into a 1.5 ml Eppendorf vial. Next, water was added to a concentration of 5 mg / ml. After preparation of the lysed sample, it was incubated at 23 ° C. for 24 hours with continuous stirring. The sample was then centrifuged to separate the precipitate from the dissolved compound. The supernatant was transferred to a labeled HPLC vial for quantification by HPLC-UV. Finally, these HPLC samples were analyzed with an HPLC-UV instrument and the amount calculated from the correction curve. The concentration of the compound in the supernatant is comparable to its solubility in water. The following water solubility was found for Bidofludimus free acid and its calcium salt.

Example 6: Determination of bioavailability The oral bioavailability of bidfludimus calcium salt and free acid was compared in Wistar rats. The free acid and calcium salt were filled into gelatin capsules and administered once to animals at a dosage level of about 10 mg free acid equivalent / kilogram body weight. Four male Wistar rats (weight range: 250-275 g) / group were treated with either bidfludimus free acid or the above calcium salt. The capsule was administered to the animal's esophagus using the application device. Venous blood samples were collected from animals under isoflurane anesthesia at the following time points after dosing: 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 32 hours and 48 hours. Clotting was inhibited using Na-heparin and plasma was obtained by centrifugation of blood samples. Plasma samples were analyzed for bidfludimus by pharmacokinetic parameters calculated by LC-MS / MS and mixed log linear trapezoidal method.

  To test the calcium salt, 4 female Lewis rats (body weight: about 200 g) / group were administered either at the dose level of 30 mg / kg (free acid equivalent) with either bidfludimis free acid or its calcium salt. I was treated. The compound was formulated in 0.5% methylcellulose phosphate buffered saline and the animals were treated by oral tube feeding. Venous blood samples were collected from animals under isoflurane anesthesia at the following time points after dosing: 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 32 hours and 48 hours. Clotting was inhibited using Na-heparin and plasma was obtained by centrifugation of blood samples. Plasma samples were analyzed for bidfludimus by LC-MS / MS and pharmacokinetic parameters (AUC) were calculated by a mixed log linear trapezoidal method.

  The oral bioavailability of the salt was observed after administration of the free acid, the area under the plasma-concentration time curve (AUC) of Bidofludimus and the mean maximum plasma concentration (Cmax value) after administration of the salt. It was evaluated by comparing with the value. These ratios are shown in Table 3 and FIG.

Example 7: Determination of long-term stability The compounds were stored at ambient temperature for 18 months (20-25 ° C., 30-60% relative humidity) and then analyzed for purity by HPLC.

Claims (6)

2- (3-Fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid calcium salt or hydrate thereof. A pharmaceutical comprising a calcium salt of 2- (3-fluoro-3'-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid or a hydrate thereof together with a pharmaceutically acceptable diluent or carrier Composition. 2- (3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid calcium salt or hydrate thereof for use as a medicament. Rheumatoid disease, acute immune disease, autoimmune disease, disease caused by malignant cell proliferation, inflammatory disease, disease caused by protozoan epidemic in humans and animals, disease caused by viral infection and Pneumocystis carinii, fiber disease, uveitis, rhinitis, asthma, and for the manufacture of a medicament for the therapy of diseases or therapeutic symptoms are selected from the group consisting of arthritis, 2- (3-fluoro-3'-methoxy - Use of calcium salt of biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid or hydrates thereof. The disease or therapeutic symptom is selected from the group consisting of graft versus host reaction and host versus graft reaction, rheumatoid arthritis, multiple sclerosis, lupus erythematosus, inflammatory bowel disease, and psoriasis. 4. Use according to 4 . A method for producing a calcium salt of 2- (3-fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid or a hydrate thereof, comprising the following:
a) adding an organic solvent suspension of calcium hydroxide to a solution of 2- (3-fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl) -cyclopent-1-enecarboxylic acid ;
b) stirring the suspension obtained in step a);
c) concentrating the at least part of the organic solvent, the suspension to give a calcium salt before asked compound;
The calcium salt of d) before asked compound, were recovered from the mixture obtained in step c); and,
washing the calcium salt before listen compound obtained in e) step d) in the organic solvent,
Including a method.

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